Project Summary/Abstract The nucleolus is a cellular organelle that carries out the synthesis of ribosomes and has a multitude of sensory and regulatory functions in the cell. Interference with biosynthetic processes in the nucleolus triggers a p53- dependent nucleolar stress response that promotes cell death in some contexts but can be cytoprotective in others, leading to increased cell resistance to genotoxic drugs. The regulatory functions of the nucleolus are thus important for understanding tumor responses to chemotherapy agents, but how exactly nucleolar stress influences therapeutic outcomes is not known. Our recent studies suggest that nucleolar stress may cross- activate components of the DNA damage response (DDR) pathway that helps to maintain genome integrity. We hypothesize that the capacity of the nucleolus to engage the DDR machinery is important for the timely activation of cellular defense mechanisms essential for cell survival. This proposal focuses on the signaling link between the nucleolus and the ATM protein kinase, a key mediator of the DDR. Using our previously developed cell models for the conditional inhibition of specific ribosome biosynthesis steps, we will determine whether ATM activation during nucleolar stress occurs in a similar or different way compared with its activation by DNA damage and oxidative stress. We will also examine chromatin markers associated with the stressed nucleolus and their possible colocalization with ATM complexes. Finally, profiling transcriptome changes in ATM-proficient and deficient cells will be used to assess the biological role of ATM in the nucleolar stress response. This study will advance our understanding of the signaling mechanisms induced by nucleolar stress. The generated knowledge will be important as it can be applied to increase the differential margins of drug sensitivity in normal and tumor cells and thus improve the efficacy of therapeutic interventions in cancer patients.